Background and Purpose: Ionizing radiation is an effective modality and widely used treatment for cancer, with over half of all cancer patients receiving radiation therapy during their course of treatment. However, despite sophisticated techniques for radiation delivery as well as the combination of radiation with chemotherapy, tumor can recur. There is now considerable evidence from both preclinical and clinical studies that the tumor recurrence can be restored following radiotherapy from influx of circulating cells consisting primarily of monocytes and macrophages. Infiltrating macrophages are a common finding in solid tumors. Tumor-associated macrophages (TAMs) can promote tumor growth and survival resulting in resistance to therapeutic treatment. While prior studies have described the monocytes/macrophage recruitment with respect to a tumor after radiation therapy, less is known about detailed interacting molecular machinery between macrophage and endothelial cells. The attachment of macrophage to endothelial cell is first step of the extravasations process. Macrophage across the vascular wall requires the sequential activation and interaction of numerous molecular effectors expressed both by endothelia cells and macrophage. In an attempt to determine the radiation specific molecular effectors of monocytes which transmigrate into the tumor, we selected nerve injured-induced protein (Ninjurin)-1. The ninj1 gene, which encodes a homophilic adhesion molecule and cell surface protein, was found to be regulated following p53 activation. More recently, Ninjurin-1 was also reported to be upregulated in inflammatory lesion, particularly on macrophages/monocytes, neutrophils, and endothelia cells. However, the exact molecules that direct the transmigration of macrophage from the blood vessel to tumors remain largely unknown.
Results and Conclusion: Endothelial cells were grown to subconfluence and irradiated. Up-regulation of Ninjurin-1 expression was measured by Western blotting and RT-PCR. Significantly, ninjurin-1 was found to be overexpressed in endothelial cell lines. Flow cytometric analysis (FACS) showed that radiation induced the surface expression of ninjurin-1 on endothelial cells. Consistent with this, we found that irradiated xenograft tumor over-expresses ninjurin-1. Radiation-induced ninjurin-1 was transcriptionally regulated by p53 and it is confirmed by transfection of p53 targeted siRNA suppressed radiation-induced ninjurin-1 expression. In addition, ninjurin-1 over-expression in endothelial cells accelerated macrophage adhesion. Adherent macrophages were counted using fluorescence microscope and FACS. On the other hand, macrophage cell line adhesion was inhibited by siRNA against to Ninjurin-1 transfection in macrophage cell line. Similar results were obtained using anti-ninjurin-1 antibodies reacting with N-terminal extracellular domain of Ninjurin-1. In an A549 xenograft model (NOD/SCID mouse) we found that irradiation induces recruitment of macrophage into the tumors. Furthermore, we found that radiation-induced macrophage infiltration in tumor was inhibited by knock out ninjurin-1 in endothelial cells. Taken together, we provide evidence that ninjurin-1 is a key molecule which produces interaction between macrophage and endothelial cells. To analyze trans association of ninjuring-1, we used fluorescence conjugated recombinant ninjurin-1. The fluorescence conjugated recombinant ninjurin-1 was interacted on irradiated endothelial cells. The recombinant ninjurin-1 protein-protein interactions were dissociated with anti-ninjurin-1 antibodies reacting with N-terminal extracellular domain of Ninjurin-1. In contrast, anti-ninjurin-1 antibody which recognizes C-terminal did not affect ninjuirn-ninjurin protein interaction and macrophage-endothelial cell adhesion. Our results show that ninjurin-1 expression on endothelial cells could increase macrophage adhesion after radiation and could be involved in the recurrence machinery action of anti-cancer radiotherapy.
Citation Format: Jong Kyu Woo, Ju-Hee Kang, Yeong-Su Jang, Goo Taeg Oh, Sang-Jin Lee, Seung Hyun Oh. Radiation potentiates the induction of macrophages infiltration into tumor by Ninjurin-1 in endothelial cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A22.
- ©2015 American Association for Cancer Research.